rna polymerases and general transcription factors final

8/13/2019 RNA Polymerases and General Transcription Factors Final

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Transcription in Eukaryotes

Eukaryote RNA polymerases and their promoters

General transcription factors in Eukaryotes

Additional bibliography: Weaver, R. F. Molecular Biology, 2nd edition (2002)


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Evidences of multiple eukaryotic polymerases

Extracts of sea urchin embryos subjected to DEAE-sephadex chromatography

Incorporated

UMP


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Cellular localisation of the three RNA polymerases

Nucleoplasma fraction

Nucleolar fraction


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Subunit structure of yeast RNA polymerase II

Epitope taggingAn epitope is genetically added to the subunit Rpb3 of the yeast pol II


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Subunit structure of RNA polymerases from Saccharomyces cerevisiae


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The three eukaryote polymerases are related to the prokaryote polymerase

(and to one another)


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The three eukaryote RNA polymerases have different roles

Studiesin vitrowith purified RNA polymerases showed that they transcribespecific regions of the genomic DNA


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Effect of-amanitin on the activity of the three eukaryte RNA polymerases

Amanita phaloides

- is a poisonous mushroom

- produces -amanitin, a potent RNApolymerase II inhibitor

%maximala

ctivity

100

50

0


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Questions that you should be able to answer

at the end of this module:

1.How many polymerases are found in Eukaryotes?

2.What type of RNA is synthesized by each of these polymerases?


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Promoters: Class I, II and III

Enhancers

Silencers

How is RNA polymerase activity iniciated?

Different polymerases have different DNA binding sites (promoters)?

How is DNA transcription regulated?


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Class I promoters

RNA pol I transcribes only one gene - the rRNA percursor gene:

Example of the human rRNA gene:

2 critical regions sensitive to mutations(upstream control element and core element)

Spacing between these regions is very important

Exists in many copies in the genome

Is variable from species to species

Different species: differences in sequence, conserved architecture

-156 -107 -45 +1 +20

UCE Core


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Class III promoters

RNA pol III transcribes short genes, that encode small RNAs:5S rRNA, tRNAs, snRNAs

5S rRNA

tRNA orVA RNA

Intermediate element

Box A Box C

Box A Box B

Internal promoters: promoter sequences are found within the genes


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Exceptions to the internal promoter in genes transcribed by RNA pol III

7SL gene

Codes for the major component of the Signal Recognition Particle (SRP)

Has a weak internal promoter and a 5flanking region is required for high-level transcription

U6 RNA gene

Has no internal promoters; the 5flancking region resemblesClass II promoters (contains a TATA box)

Homework:What is the SRP?

Homework:Function of U6 RNA?


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Most common element in the promoteris the TATAAA consensus sequence

Class II promoters

Present in genes that code for proteins and snRNA genes

TATA box

Other TATA box sequences may be found due to sequence variation

T frequently replace A

G and C can substitute nucleotides in the TATA box

example: the rabbit-globin gene promoter starts with CATA

GATA is another variation found in class II promoters


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Variable location;

In yeast, it can be found from50 to 70 bp upstream thetransciption start site

Class II promoters: core promoter + upstream element

TATA box (positioned around -25 nt )Core promoter Initiator, centered on the transcription start site

Downstream element


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What is the function of the TATA box?

Effects of deletions in the histone H2A promoter of the sea urchin

Specialised genes have TATA boxes

Genes that are expressed only in certain types of cells (ex: keratin in skin

cells; hemoglobin in red blood cells) or in certain environmental conditions

Removing the TATA boxcauses transcription to initiateat a wide variety of sites

A B C

TATA


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The TATA box is also involved in positioning the transcription start site

Effect of deletions in the SV40 (simian virus 40) promoter:

The distance between the TATA box and the transcription initiation siteremained constant

Transcription started about 30 bp downstream of the first T of the TATA box (usually with a purine)


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In some promoters, removal of the TATA box impairs promoter function

Removal of the TATA box of the rabbit-globin gene inhibits transcription

Wtfragment

Shorterfragment

1

2

3

wt


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Molecular Biology technique 1: S1 Nuclease Protection Assay

Probe molecules or RNA moleculesthat do not hybridize are removed byS1 nuclease digestion

The hybridmolecules areethanol-precipitated

Recovered hybridmolecules areseparated on adenaturingpolyacrylamide-gel followed byautoradiography

The size and abundance of the protected moleculesis a direct measure of the steady-state level for a specific RNA


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Molecular Biology technique 2: Primer extension

Transcription

Hybridize labeled primer

Extend primer with reverse transcription

DNA

RNA

Denature hybridElectrophoresis


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Genes transcribed by RNA Pol II may not have TATA box promoters

Constitutively active. Found in housekeeping genesTranscription is regulated by Sp1 (Specific protein 1)Ex: adenine deaminase, part of the nucleotide synthesis pathway

TATA-less promoters

- GC boxes: GGGCCC or CCGCCC

- CCAAT box

- Other initiator sequences

Homeotic genes (regulated during development)

Ex:Genes that control the development of the fruit fly, or genes activated during the developmentof the immune system in mammals. Ex:TdT gene (mouse terminal deoxynucleotidyl transferase)contains a 17 bp initiator sequence sufficient to drive basal transcription

Transcription is regulated byCCAAT-binding transcription factor (CTF)and CCAAT-enhancer binding protein (C/EBP)Ex: Herpes simplex virus (HSV) timidine kinase promoter

HSV


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Some need the TATA box for proper functioningEx: rabbit -globin gene promoter

Some need the TATA box for correct positioning of the Pol II

at the transcription start siteEx: SV40 (simian virus 40) promoter

Some dont have a TATA box and are functional

(TATA-less promoters)Ex: GC boxes and CCAAT box

Important concepts regarding Class II promoters


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Transcription initiation depends on the formation of a protein complex

The pre-initiation complex

In Class II promoters

The preinitiation complex is assembled at the TATA box position

Assembly is mediated by TBP, which recognises the TATA boxand allows the condensation of other protein factors

TBP-associated factors, TAFs or TAFIIs

A large multi-component complex in

which RNA polimerase interactswith accessory proteins known asthe general transcription factors(GTFs)


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Molecular model of the TATA box-binding protein (blue and purple)complexed with DNA (pink).

The TATA box is recongnised by TBP - TATA box Binding Protein

- Is highly conserved in eukaryotes

-Binds to the minor groove

of the TATA box


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Structure and composition of TFIID

Hypothetical structure of thereconstituted DrosophilaTFIID

Gel electrophoresis of in vitroreconstituted TFIID from Drosophila.

Eight major TAFIIs are visible in thegel.

TFIID composition is highlyconserved from yeast to man


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How is the preinitiation complex formed in TATA-less promoters?

TBP association with the TATA box

Complex with all the TAFII factorsthat form TFIID

Complex with a sub-set of TAFII factors

TAFII250 and TAFII150 are sufficient to

bring TBP to the Initiator sequence


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TFIID association with GC box promoters

in the complete TFIID complex

The complex formed byTFII250, TFII150 and TFII150is sufficient to anchor TBP to Sp1.

TAFII110 interacts with Sp1


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Different TAFII combinations respond to different activators

TAFII250 and TAFII110 promote the

interaction between TFIID and Sp1,the protein that binds to GC boxes

TAFII250 and TAFII150 mediate

the association of TFIID to the

Initiator sequence and to

downsteam elementsTAFII250 is an histone acetyl-

transferase and a kinase;

modifies histones and other GTF


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A model for transcripton enhancement by activators

TFIID supports activation by several activators


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TATA

Start

Downstreamelement

GCbox

CCAATbox

Recognisedby Sp1

Recognised byCTFC/EBP

The rate of transcription is regulated by promoter proximal elements

Trans-acting elementsactivator proteins (DNA binding domain + activator domain)

Stabilize the preinitiation complex, increasing the frequency of transcription


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GC boxes may also work as transcription stimulators

- Orientation-independent, like enhancersthey can be flipped 180 and still function

- Position-dependent, like other promotersif they are moved more than a few base pairs away from the TATA box they loose theability to stimulate transcription

A specific transcription factor, known as

Specificity Protein 1 (Sp1)

binds to the GC boxes and stimulates transcription

GC boxes are

Sp1


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Tissue specific effectThey depend on the activity of DNA binding proteins that are specific to a given tissue

Enhancers - stimulate

Silencers - repress

Silencers may partially or completely repress transcription

High levels of transcription; enhancers work in either orientation

They are position and orientation independent

unlike promoters, which are position and orientation dependent!

Cis-acting elements: distal DNA elements that strongly influence transcription


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TFIID functions

Recognition of DNA sequence elements of the core promotermay be mediated directly by some TAFIIs or may be activator-dependent(Sp1 mediates the link to GC boxes)

Generation of a chromatin environment favourable for transcription initiation

Induces structural modifications in other general transcription factors, facilitating the assembly

of the pre-initiation complex and transcription initiation


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Several GTPs compose the pre-initiation complex

Factor Number ofsubunits

MW(kD)

Function

TBP

TFIID-TAFs

TFIIA

TFIIB

TFIIF

TFIIE

TFIIH

1

12

3

1

2

2

9

38

15-250

12, 19, 35

35

30, 74

34, 57

89, 80, 62,52, 44, 34,32, 38, 40

Recognize core promoter

Recruit TFIIB

Assist transcription activationAssist promoter recognition

Stabilize TFIID and promoter binding

Recruit RNA Pol II and TFIIF

Assist RNA Pol II to reach promoter

Promoter meltingRecruit TFIIH and modulate its helicase,ATPase and kinase activities

Phosphorilation of the CTD of RNA pol IIPromoter melting using helicase activity


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Formation of the DABPolFEH pre-initiation complex(from in vitroexperiments)

TFIID + TFIIA + TFIIB

DAB complex is assembled on the TATA box(TFIIA is optional in vitro)

1)

2) TFIIF binds to polymerase and leads it to the DAB complex

DABPolF complex

3)

E

H

DABPolFEH complex

Finally, TFIIE and TFIIH join the DABPolF complex

DABPolF + TFIIE + TFIIH

DAB complex


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General transcription factor requirements in Yeast


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Factors involved in the regulation of transcription by RNA Pol II

Long regions of the DNA can loop over to enable the regulatory connections


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at the end of this module:

1. Which are the main elements of Class I, II e III promoters? Where are they localized?

2. What is the function of the TATA box in class II promoters? Which protein recognises the TATA box?

3. How is a pre-initiation complex formed in class II promoters that have TATA box?And in promoters that dont have a TATA box?

4. What is the function of the GC box in class II promoters? Which protein recognises the GC box?

5. Beyond TATA boxes and GC boxes, what other motifs can be found in class II promoters?

6. What is the function of the Initiator in class II promoters?

7. What are proximal activator sequences? Where are they located and how do they work?

8. What are enhancer and silencer sequences? Where are they located and how do they work?

9. What is the DABPolFEH complex? What is the function of the DABPolFEH complex?

10. What is the function of the general transcription factors TFIIB, TFIID, TFIIF, TFIIE and TFIIH?


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Stabilises TFIID

in the promoter

Links TFIID and TFIIF/Pol II,promoting the assembly of thecomplete pre-initiation complex

Directs RNA Pol II to promoterswith assembled DAB complex

Reduces non-specific interactions

between RNA Pol II and the DNA

TFIIH

TFIIE

recruits TFIIHzinc finger domain binds to DNA

TFIIF

Phosphorylation of RNA Pol II

Promoter melting throughhelicase activity

TFIIA

TFIIB

A model for the pre-initiation complex


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TFIIS stimulates RNA Pol II proofreading of transcripts


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TFIIS stimulates RNA Pol II proofreading of transcripts

Incorrect nucleotides (in yellow)may be incorporated by RNA Pol II

RNA Pol II backtracks, extrudingthe 3 end from the active site

The RNAse activity of the RNA Pol IIeliminates the incorrect nucleotide

RNA Pol II resumes activity

Th RNA P l II h l


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Core

Holoenzyme

The RNA Pol II holoenzyme

The RNA Pol II holoenzyme is a complex formed byRNA Pol II and a subset of initiation factors, found notlinked to the promoter

In yeast, the RNA Pol II holoenzyme contains a subset of andregulatory proteins (SRB/mediator proteins) and generaltranscription factors, including TFIIF

The composition of the holoenzyme depends onthe type and physiological state of the cell

In mouse liver cells, the RNA Pol II holoenzyme containsTFIID, B, E and F

Preinitiation complex formation at Class I promoters


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Preinitiation complex formation at Class I promoters

SL1 interacts with the RNA Pol Ito strengthen the binding of thecomplex to the UCE

UBF stimulates transcriptionthrough interactionwith the UCE

TFIA is essentialfor RNA Pol Irecruitment


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TFIIIA is a zinc-finger protein


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http://www.youtube.com/watch?v=GRL_rdB30GY&feature=related

Homework:

video on YouTube

It is composed of 9 zinc-fingers in tandem repeatinteracts with the DNA through the major groove

Zinc-finger structural motif-helix

-sheetZinc

The order of binding of transcription factors is important


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5S rRNA

The order of binding of transcription factors is important

5S rRNA gene transcription only occurres whenTFIIIB is added after TFIIIA and TFIIIC

TFIIIB cannot bind to the promoter by itself;only binds if TFIIIC and TFIIIA are already present


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Molecular Biology technique 3: DNA footprinting

DNA containing a transcription factor binding site

Transcription factor

Radioactive label

footprint

fragments

Electrophoresisand autoradiogram

Finding the DNA sequence targeted by a DNA binding protein

DNAse I digestion under mild conditions(approximately 1 cut per DNA molecule)

TFIIIB and TFIIIC DNA binding sites in a tRNA gene


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TFIIIB and TFIIIC DNA binding sites in a tRNA gene

DNA footprinting using a labeled tRNA gene andcombinations of purified TFIIIB and TFIIIC

In d, when the sample was treated with heparin to

strip off loosely bound protein, TFIIIB remainsbound to the upstream region but TFIIIC wasremoved

In b, TFIIIC protects the internal promoter, especiallyBox B, but not the upstream region

In c, when TFIIIC and TFIIIB are present, theupstream region is also protected

TFIIIC

TFIIIB

TFIIIC

TBP plays a role in the formation of the preinitiation


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complex in Class III promoters

TFIIIC organizes the formation of thepreinitiation complex, promoting thebinding of TFIIIB (with TBP) in theupstream region

TFIIIB promotes the binding of RNAPol III at the Start site

As RNA Pol III proceeds, TFIIIC isremoved but TFIIIB remains in place

TFIIIB is composed of TBP andtwo TAFs: TAF-172 and TAF-L


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yat the end of this module:

1. O que o complexo DAB? Qual a sua funo?

2. Como constitudo o complexo DABPolFEH?

3. Qual o significado funcional da actividade de acetil-transferase de histonas e de cinase de TAFII250?

4. Nos promotores sem TATA como que se liga o complexo TFIID ao promotor?

5. Qual a funo das caixas GC na ligao do complexo TFIID ao promotor?

6. Qual a funo de TFIIA e de TFIIB na formao do complexo de pr-iniciao? e qual a funo dosfactores TFIIF, TFIIE e de TFIIH?

7. Qual o significado funcional da fosforilao da terminal carboxlico da RNA polimerase II (CTD)?

8. Qual a funo dos factores de alongamento da transcrio?

9. Quais os factores gerais da transcrio dos genes da Classe-I?

10. Qual a funo e como constitudo o complexo SL1?

11. Qual a funo do complexo UBF? Com o funciona?

12. Quais so os factores gerais da transcrio dos genes da Classe-III?

13. Como funcionam os factores TFIIIA, TFIIIB e TFIIIC?

14. Qual a funo da TBP na formao do complexo de pr-iniciao das classes I, II e III?

rna polymerases and general transcription factors final

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